Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian Gycogen Synthase Kinase-3 is also known as GSK3. GSK3, which has alpha and beta isoforms, can phosphorylate serine and threonine residues in protein or peptide substrates. GSK3 also autophosphorylates itself on a tyrosine residue. Some, but not all, in vivo GSK3 phosphorylation events depend upon prior phosphorylation of the substrate by another cellular kinase. The enzymatic activity, activation and regulation of GSK3 have been studied. Many cellular substrates of GSK3 have been identified. Furthermore, polypeptides have been studied to examine GSK3 substrate specificity. While polypeptides and variants thereof have been studied as individual GSK3 substrates or ligands, mixed ligands linked together as polyligands that modulate GSK3 activity have not been demonstrated before this invention. An aspect of the invention is to provide novel, modular, inhibitors of GSK3 activity by modifying one or more natural substrates either by truncation or by amino acid substitution. A further aspect of the invention is the subcellular localization of a GSK3 inhibitor, ligand, or polyligand by linking to a subcellular localization signal.
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Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also U.S. Pat. No. 7,071,295.